Comparison of the Effect of Velvet Antler from Different Sections on Longitudinal Bone Growth of Adolescent Rats

The aim of this study was to compare the effectiveness of velvet antler (VA) from different sections for promoting longitudinal bone growth in growing rats. VA was divided into upper (VAU), middle (VAM), and basal sections (VAB). An in vivo study was performed to examine the effect on longitudinal bone growth in adolescent rats. In addition, in vitro osteogenic activities were examined using osteoblastic MG-63 cells. VA promoted longitudinal bone growth and height of the growth plate in adolescent rats. Bone morphogenetic protein-2 (BMP-2) in growth plate of VA group was highly expressed compared with control. The anabolic effect of VA on bone was further supported by in vitro study. VA enhanced the proliferation, differentiation, and mineralization of MG-63 cells. The mRNA expressions of osteogenic genes such as collagen, alkaline phosphatase, and osteocalcin were increased by VA treatment. These effects of in vivo and in vitro study were decreased from upper to basal sections of VA. In conclusion, VA treatment promotes longitudinal bone growth in growing rats through enhanced BMP-2 expression, osteogenic activities, and bone matrix gene expressions. In addition, present study provides evidence for the regional differences in the effectiveness of velvet antler for longitudinal bone growth.


Introduction
Longitudinal bone growth, which determines the size and shape of the body frame, proceeds at a rate distinct from that of muscle and other tissues and is controlled by specific mechanisms. During childhood and adolescence, the long bones grow at the ends of the bones which occur at the growth plate by endochondral ossification of epiphyseal plate [1]. Growth plate chondrocyte proliferation and hypertrophy lead to formation of new cartilage, chondrogenesis, and remodeling of the newly formed cartilage into bone tissue resulting in longitudinal bone growth [2].
The current methods for increasing final height include treatment with a growth hormone alone or in combination with an analog of gonadotropin-releasing hormone [3]. However, it is generally too expensive and also has harmful side effects such as prepubertal gynecomastia, arthralgia, edema, benign intracranial hypertension, insulin resistance, and leukemia [4]. On that account, there has been an increasing awareness of the benefits of natural products, and many plant-derived medicines have been reported. However, the researches on animal medicines or functional foods are still relatively few, although animal medicines have been proven of various important components, such as proteins, peptides, fatty acids, glycosaminoglycans, prostaglandins, vitamins, minerals, dietary fiber, essential oils, and carotenoids [5,6], which can be used in the prevention and treatment of various diseases.
Velvet antler (VA), the immature antler of male deer, is traditionally used for thousands of years in Asian countries, such as Korea, China, Taiwan, and Mongolia. It is currently estimated that the global production of velvet antlers is near to 1300 tons/year, which is still rapidly growing to meet the requirements of medicinal markets [7]. As a typical traditional animal medicine, VA has pharmacological effects to improve immune system, physical strength, and sexual function [7,8]. VA, also, has been reported to possess bone strengthening activity and has been used in treating arthritis, osteoporosis, and fracture in animal model or human clinical trials [9][10][11][12].

Evidence-Based Complementary and Alternative Medicine
A whole VA stick is usually divided into four portions, with the value decreasing from the tip to the base. Chemical analyses of VA revealed that there are regional differences in chemical composition: the contents of proteins and lipids decrease downward from the tip to the base, while those of ash, calcium, and collagen increase [13] suggesting the degree of calcification increased from tip to base section of VA. During the ossification of deer antler, the total collagen content was found to be increased. Typically, the antler is cut off near the base after it is about two-thirds of its potential full size, between 55 and 65 days of growth, before any significant calcification occurs. Traditionally, the market values of antlers are downgraded with increasing degree of calcification. However, which parts of VA are suitable for preventing and managing bone health especially bone growth had not been clarified.
The objective of the present study was to compare the effect of VA from different sections on longitudinal bone growth of adolescent rats and elucidate the underlying mechanisms for the effect.

Materials and Methods
2.1. Materials. VA, obtained from farmed Elk deer 75 days after casting, was kindly provided by the Animal Genetic Resources Station (National Institute of Animal Science, South Korea). VA was divided into upper (VAU), middle (VAM), and basal (VAB) sections ( Figure 1). The criteria used for dividing the antler into three sections is defined as follows: upper section, top 15 cm of the part of the main beam to the 2nd division of a deer's antlers from its head; middle section, the upper half of the remaining main beam; and base section, the lower half of the remaining main beam. Total part of each section was sliced with a bone slicer, freeze-dried, and ground into powder. The powdered VA was immersed in 70% ethanol for 3 days, filtered, concentrated by vacuum evaporation, and finally subjected to freeze drying. Respective yields of sections, from upper to basal, were 3.4% (VAU), 2.9% (VAM), and 1.7% (VAB).

Animals.
Longitudinal bone growth was determined in 3-week-old male Sprague-Dawley rats (Samtako Co., Osan, Korea). The experimental procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee, Semyung University (smecae 15-09-01). The animals were housed under controlled temperature and lighting conditions. The rats were randomly divided into four groups ( = 8); vehicle (control, distilled water) and upper (VAU), middle (VAM), and basal (VAB) sections of antler extract (100 mg/kg) were orally administered daily via stomach tube for 5 consecutive days. Treatment dose was decided from the preliminary study. On the sixth day, all rats were sacrificed under chloral hydrate anesthesia for tissue analysis.

Measurement of Longitudinal Bone Growth.
To measure the effect on longitudinal bone growth rate, calcein was used as a fluorescence marker to label the bone line on the surface of the tibia. Calcein plays the role of fluorescent dye under ultraviolet illumination. Calcein (10 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) was injected intraperitoneally 24 h before sacrifice. The dissected tibias were fixed in 0.1 M phosphate buffered formalin for 2 h, decalcified, and dehydrated by immersing in 30% sucrose for 1 day at 4 ∘ C. Dehydrated bone was sectioned longitudinally at sagittal sections of proximal part with a thickness of 40 m using a sliding microtome (HM440E, Zeiss, Germany). Bone growth was measured by measuring the gap between fluorescent line formed by calcein and the epiphyseal end line of the growth plate at three different locations using a fluorescent microscope (BX60, Olympus, Tokyo, Japan), and the averages were obtained.

Measurement of Growth Plate
Height. Cresyl violet staining (Sigma-Aldrich) was used to stain the chondrocytes in the growth plate of the samples. Each tibia sample was sectioned longitudinally at a thickness of 40 m using a sliding microtome as described above and stained with cresyl violet. The growth plate height was measured at three different locations and the averages were obtained.
2.6. Cell Culture. Human osteoblast-like MG-63 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured at 37 ∘ C in a 5% CO 2 atmosphere in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, MD, USA) containing 10% heat-inactivated  2.8. Alkaline Phosphatase Activity. Alkaline phosphatase (ALP) activity was measured using enzymatic assay. MG-63 cells, incubated in DMEM, were seeded in 12-well plates (50,000 cells/well) and incubated for 24 h. The medium was changed to osteogenic medium (DMEM with 10 mM -glycerophosphate, 5 nM dexamethasone, and 50 g/mL ascorbic acid); VA samples (50 and 100 g/mL) were added to the cells and incubated for another 24 h. The 50 and 100 g/mL concentrations were chosen since the maximum effect and plateau had been gained under those two concentrations in cell proliferation assay results. The cells were then rinsed with PBS and lysed in 300 L of 0.2% of Triton X-100. The cell lysates were centrifuged at 13,000 rpm for 5 min. The supernatant of the lysate was used for the measurement of ALP activity by measuring the release of p-nitrophenol from p-nitrophenylphosphate at pH 9.8.

Collagen Content.
Collagen synthesis was measured using picrosirius red method [14]. MG-63 cells were seeded in 12-well plates (50,000 cells/well) and allowed to adhere overnight. The medium was changed to osteogenic medium; VA samples (100 g/mL) were added to each well and incubated for 7 days. Cells were fixed with Bouin's fluid and stained with 0.1% Sirius red (Direct Red 80, Sigma-Aldrich) in a saturated aqueous solution of picric acid for 30 min. Stained dye was dissolved and the absorbance was measured at 540 nm.

Calcium Deposition.
The formation of calcium phosphate was determined by alizarin red-S assay [15]. MG-63 cells were seeded and incubated as described above in the collagen assay process. After 7 days, cells were fixed with 10% formaldehyde and stained with 2% of alizarin red-S (pH 4.2, Sigma-Aldrich) at room temperature. The stained alizarin red-S was extracted with 10% acetic acid, and the amount of calcium deposition was quantified using the absorbance of extracted alizarin red-S at 405 nm.

Real-Time PCR.
Real-time PCR was performed to analyze relative gene expression. MG-63 cells were seeded in 6-well plates (100,000 cells/well) and allowed to adhere overnight. The medium was changed to osteogenic medium; VA sample (100 g/mL) was added to each well and incubated for 24 h. Total RNA was extracted using RNeasy5 Protect Mini Kit (Qiagen, Valencia, CA, USA), and cDNA was synthesized from mRNA using QuantiTect5 Reverse Transcription Kit (Qiagen). Real-time PCR was performed using QuantiTect6 SYBR5 Green PCR Kit (Qiagen) according to the manufacturer's protocol. The PCR primer sequences are shown in Table 1. Analyses were performed using Rotor-Gene Q5 (Qiagen) and gene expression values were calculated based on the comparative ΔΔCT method according to the manufacturer's protocol.

Osteoclastogenesis Assay.
For induction of osteoclastogenesis, osteoclast precursor cells prepared as described above were further cultured for 4 days with RANKL (50 ng/mL) and M-CSF (30 ng/mL) in the presence of VA samples. Osteoclasts were identified by staining tartrateresistant acid phosphatase (TRAP), a marker enzyme of osteoclasts [16], using commercial kit (TRAP 387-A Kit, Sigma-Aldrich). TRAP-positive multinucleated cells with ≥3 nuclei were counted as osteoclasts.

Statistical
Analysis. The data were expressed as mean ± SD. One-way ANOVA followed by Tukey's multiple comparison test was performed for statistical analysis (GraphPad Prism ver. 6), and values of less than 0.05 ( < 0.05) indicated significant differences. All in vitro experiments were performed with triplicate independent samples.

Effects on Cell Longitudinal Bone Growth.
To determine the growth per day, instead of the total growth over a period, exact methods for measurement are necessary. Recently, the use of tetracycline or calcein as intravital markers of the growth process to label mineralizing bone in the rat has been reported [17,18]. In the present study, calcein was used to label newly formed bone for the determination of longitudinal bone growth. Calcein binds to free calcium and gets deposited in newly deposited bone, causing staining and fluorescence under ultraviolet illumination. The effects of the different sections of VA on the rate of longitudinal bone growth from the proximal tibia in the rat are depicted in Figure 2. The fluorescent line corresponds to the injection of calcein which binds with calcium in the newly formed bone (Figure 2(a), lower arrow). The arrow between the fluorescent line formed by calcein (Figure 2(a), lower arrow) and epiphyseal end line of the growth plate (Figure 2(a), upper arrow) indicates the length of the bone growth during 24 h period. Longitudinal bone growth was significantly increased by VAU treatment compared with the control (Figure 2(a)). Figure 2( growth, statistical significance was not observed. The present study provides first evidence for the effectiveness as well as regional differences of VA on longitudinal bone growth in adolescent rats. Recently, Tseng et al. [19] compared the antiosteoporotic activity of VA from different sections using ovariectomyinduced osteoporosis animal model and suggest that the upper and middle sections of VA were equally effective in protecting bones from an estrogen-deficient state. However, the effects on proliferation and mineralization of osteoblasts, MC3T3-E1 cells, were decreased downward from upper section to the base [19] supporting the results of the present study. In addition they reported that the level of insulinlike growth factor 1 (IGF-1) was decreased downward from upper section to base section. Longitudinal bone growth is a function of growth plate chondrocyte proliferation and hypertrophy, and IGF-1 is reputed to augment longitudinal bone growth by stimulating growth plate chondrocyte proliferation [20]. Therefore, these data support the results of the present study, and one of the possible mechanisms may be explained by the IGF-1 level in each section of VA.

Effects on Growth Plate
Heights. The growth plate, which is located at the distal end of the bone, is the main location where longitudinal bone growth occurs owing to the stimulation of chondrocyte proliferation. The growth plate consists of four distinctive histological zones beginning with the resting zone and extending through the proliferative, hypertrophic, and ossification zones (Figure 3(a)). The topmost layer, the resting zone, contains chondrocytes that serve as stem-like cells for the growth plate, with the potential to generate clones of rapidly proliferating chondrocytes, which are located in the proliferative zone in the second layer of the growth plate [21]. The proliferative zone is the driving force behind bone elongation. Over time, as longitudinal growth proceeds, proliferative cells close to the hypertrophic zone undergo terminal differentiation. During this process, they stop proliferating and physically enlarge to become hypertrophic chondrocytes, composing the third, hypertrophic layer of the growth plate. In the ossification zone, the chondrocytes eventually die and are transformed into bone matrix where longitudinal bone growth occurred [22]. Since the synchronized processes of chondrocyte proliferation and cartilage ossification in growth plate lead to longitudinal bone growth [23], the rate of longitudinal bone growth is regulated by the rate of chondrocyte proliferation of the growth plate [24]. In the present study, the rate of chondrocyte proliferation was determined by measuring the height of the proximal tibia growth plate (Figure 3(a)). The height of the growth plate in the control group was 511.2 ± 21.6 m, and administration of 100 mg/kg VAU and VAM significantly increased the height of the growth plate to 567.4 ± 23.4 m and 566.2 ± 20.6 m, respectively (Figure 3(b)). The effect of VAU (11.0%) was similar to that of VAM (10.8%), while VAB caused 6.3% increase which was not statistically significant compared with control. These results suggest that the effect of VAM was slightly lower but not statistically significant compared with VAU, and the effect decreases downward from upper section to the base.

Effects on BMP-2 Expression.
BMPs play important roles in regulating growth plate chondrogenesis and longitudinal bone growth. Of the various forms of BMP, BMP-2 plays an important role in the development of the epiphyseal growth plate [25]. BMP-2 stimulates chondrocyte proliferation in the proliferative zone of the growth plate and also causes an increase in chondrocyte hypertrophy [26]. In this context, it has been suggested that alterations in the expression or production of BMP-2 can modulate the proliferation and activity of bone forming cells.
In the present study, protein expression of BMP-2 in growth plate was highly expressed in hypertrophic and ossification zones compared to resting and proliferative zones (Figure 4(a)). As expected, BMP-2 expressions particularly in hypertrophic and ossification zones of the growth plates were increased in the VAU and VAM treated groups compared with the control group (Figures 4(a) and 4(b)). Numerical values of the BMP-2 expression were increased by 25.8%, 24.7%, and 10.5% with VAU, VAM, and VAB treatment, respectively, compared with control (Figure 4(b)). The effect on BMP-2 expression in growth plate, which is related to the bone growth and formation, was also decreased downward from upper section to the base. Furthermore, the effect of VAM was similar to VAU. The results of the in vivo experiment suggest that treatment with VA increases longitudinal bone growth rate by promoting chondrocyte proliferation and chondrogenesis through the upregulation of BMP-2 expression in growth plate, and the effect decreases downward from upper section to the base.

Effects on MG63 Cell
Osteogenesis. Bone regeneration is regulated by a fine balance of biochemical and cellular events that ultimately stimulate osteoblasts to produce new tissue, in particular, new extracellular matrix composed mainly of collagen. The collagen matrix is then mineralized via ALP activity, which induces formation of calcium phosphate crystal seeds. Bellows et al. [27] reported that ALP is one of the osteoblast phenotype markers and an essential enzyme for mineralization. Hence, osteoblast proliferation, collagen content, and ALP activity, as early differentiation markers, and cellular calcium content, as a late marker of differentiation, were examined to investigate the effects of VA on the differentiation of MG-63 cells. First, the effect of different sections of VA on osteoblast proliferation was examined by BrdU incorporation. As shown in Figure 5(a), MG-63 cell proliferation was significantly and dose-dependently increased with VAU and VAM treatment ( < 0.05). MG-63 cell proliferation reached peak value at 50 g/mL VAU treatment exhibiting 119% of the basal value, while VAM treatment reached peak at 100 g/mL exhibiting 111% of the basal value. VAB at the highest concentration (100 g/mL) significantly ( < 0.05) increased cell proliferation compared to the control exhibiting 106% of the basal value. Effects of VAU and VAM on increasing cell proliferation were significantly higher than those of control at 50 and 100 g/mL concentration. Therefore, the effect of VA on ALP activity in MG-63 cells was further determined at 50 and 100 g/mL concentration. VAU treatment significantly increased the ALP activity, an early-stage osteoblasts differentiation marker, at 100 g/mL concentration ( < 0.05, Figure 5(b)) compared with control. VAM and VAB failed to affect ALP level. Hence, collagen content ( Figure 5(c)) and calcium deposition ( Figure 5(d)) were determined at 100 g/mL of VA concentration. Collagen synthesis was significantly increased with VAU (11.2%) and VAM (6.2%) treatment compared with control, and the increased collagen synthesis was significantly higher than VAB. Cellular calcium deposition was examined using Alizarin red-S staining. Significant increases in mineralization were found with VAU and VAM treatment compared to the control and VAB group. Mineralization was significantly increased to 118% and 107% of the control by VAU and VAM treatment, respectively. The anabolic effect of VA on bone was further supported by the findings in this study demonstrating that VA enhanced the proliferation, differentiation, and mineralization of osteoblastic cells. Furthermore, the effects of VAU were greater than the effect of VAM, suggesting that the effects were decreased from the upper section to the base section. Lee et al. [28] reported that VA water extract enhanced osteoblasts proliferation and mineralization. Furthermore, Tseng et al. [19] reported that VAU and VAM dose-dependently increased osteoblasts proliferation as well as mineralization supporting the results of the present study.

Effects on Osteogenic Gene Expression.
It is known that preosteoblastic cells produce proteins of the extracellular matrix including collagen at first and then to successively produce alkaline phosphatase (ALP) and osteopontin/osteocalcin during mineralization phase [29]. Accordingly, there is increased expression of osteogenic genes in osteoblasts during the bone formation process, and these genes play roles in extracellular matrix formation and mineral deposition. Collagen (COL) is a primary gene product of osteoblasts during bone matrix formation and comprises 85-90% of the total organic bone matrix [30]. Noncollagenous matrix proteins also have a great importance in regulating of ossification and bone remodeling. The most abundant noncollagenous protein produced by osteoblasts is osteocalcin (OCN), a phosphorylated glycoprotein which has high affinity for binding ionic calcium and physiologic hydroxyapatite [31]. Osteopontin (OPN) is an osteoblast-derived, heavily glycosylated protein of the bone matrix, which is expressed at late stages of differentiation, and its appearance closely correlates with the appearance of mineral [31]. In order to examine the anabolic effect of VA, the mRNA expressions of COL, ALP, OCN, and OPN in MG-63 cells were measured by real-time PCR ( Figure 6).

Effects on Osteoclastogenesis.
Osteoclasts are the only cell type capable of resorbing mineralized bone. To examine the effect of VA on osteoclast differentiation, bone marrow-derived osteoclast precursor cells were cultured in osteoclastogenic media with different parts of VA extract. TRAP-positive multinuclear osteoclasts were generated in response to stimulation from M-CSF and RANKL. The results showed that the addition of VA did not affect osteoclast formation (data not shown). Bacillus-fermented VA extract has been reported to inhibit osteoclast differentiation [32] which does not support the results of the present study. However, Lee et al. [28] suggested that VA, fermented with Cordyceps militaris, contain more sialic acid than nonfermented VA, and the stimulatory effects on osteoblastic cell proliferation and ALP production were increased by fermentation. Therefore, the effect of nonfermented VA in the present study may not be strong enough to induce osteoclast differentiation.

Conclusions
In conclusion, results of the present study suggest that VA promotes longitudinal bone growth in adolescent rats through, at least in part, MG-63 cell osteogenesis, and the effect was decreased downward from upper section to basal section. VA stimulated the proliferation, differentiation, and mineralization of osteoblasts through upregulation of osteogenic gene expressions. These results support the stimulating nature of VA toward the function of osteoblastic cells. Although the molecular mechanisms underlying osteoblast differentiation remain to be defined, the results of the present study suggest that BMP-2, which plays an important role in regulating osteoblast differentiation and subsequent bone formation, might participate in these mechanisms. Furthermore, the present study provides strong evidence for the regional differences in the effectiveness of VA in longitudinal bone growth. However, further studies are needed to elucidate the bioactive chemical constituents associated with these effects.
The present study has some limitations. Only a single dose of VA was used in the in vivo study. The dose for traditional use of VA is usually 1 g/day, taken all at once or divided throughout the day. Therefore, the dose of 100 mg/kg/day given to the experimental rats in this study was equivalent to the traditional human dosing regimen of 1 g/day based on body surface area conversion, where animal dose is equal to human equivalent dose × 6.2, assuming that an adult person's weight is 60 kg [33].